In eukaryotes, cyclin B-bound Cdk1 promotes mitotic entry, but is held in check, in part, by the protein kinase, Wee1. In budding yeast, inactivation of Swe1 (Wee1 homologue), which directly phosphorylates and negatively regulates Cdc28 (Cdk1 homologue), is required for timely mitotic entry. During an unperturbed cell cycle, Swe1 accumulates in S phase and becomes sequentially hyperphosphorylated, generating several tiers of multiple isoforms, and then undergoes ubiquitin-mediated degradation largely by the ubiquitin ligase (E3) known as the anaphase-promoting complex (APC). However, despite the homology of Hsl1 to S. pombe Nim1/Cdr1 within its catalytic domain and beyond, and contrary to the reported phosphorylation of S. pombe Wee1 by recombinant Nim1/Cdr1, Hsl1 does not directly phosphorylate Swe1. We found that multi-step phosphorylation of Swe1 is mainly achieved by two distinct kinases, Cla4 (a PAK homologue) and Cdc5. Cla4-dependent Swe1 phosphorylation occurs in S phase, when it localizes to the incipient bud site, whereas bud-neck-localized Cdc5 activity is required for Swe1 phosphorylation in M phase. Both of these events are critical for timely degradation Swe1. In addition, mitotic Clb2-associated Cdc28 activity, but not G1 or S-phase Cdc28 activity, directly phosphorylates Swe1 in vitro. Consistent with this observation, acute inactivation of a chemically-sensitive cdc28-as mutant (data not shown) or depletion of mitotic cyclins (Clb1 - Clb4) results in stabilization of Swe1 in vivo. These data suggest that, in addition to Cla4 and Cdc5, Clb-Cdc28 activity is required for proper Swe1 regulation. One intriguing model is that recruitment and activation of Cla4 at the incipient bud site may be a hallmark that indicates that proper morphogenesis is underway, whereas recruitment of Cdc5 to the bud-neck perhaps signals relief from the Swe1-imposed checkpoint. Finally, Clb-Cdc28-mediated phosphorylation of Swe1 could act as a positive feedback mechanism for the final stages of Swe1 inactivation. Another possibility is that Cla4, Cdc5, and Clb-Cdc28 are required in sequential fashion because prior phosphorylation by one kinase is necessary for efficient phosphorylation by the next. Thus, it is likely that Swe1 phosphorylation represents a nodal point for integrating signals from multiple kinases (Cla4, Cdc5, and Clb-Cdc28) that license passage into mitosis. To investigate the molecular mechanisms as to how these three kinases contribute to the temporal and spatial regulation of Swe1 during the cell cycle, we plan to carry out both genetic and in vivo biochemical experiments. In vitro reconstitution experiments will be carried out to examine whether Swe1 phosphorylated by these kinases in an ordered manner is susceptible to APC-dependent ubiquitination. In collaboration with Dr. T. Veenstra's group in NCI-Frederick, experiments are also under way to map all the in vivo Swe1 phosphorylation sites using nanoelectrospray tandem mass spectrometry. Comparison between the in vivo phosphorylation sites with those of in vitro sites will be conducted to dissect the kinase(s) responsible for each phosphorylation event. These collective studies may likely provide new insights as to how multiple kinases coordinate to achieve hyperphosphorylation and thereby timely degradation of Swe1 prior to onset of the G2/M transition.